Method for generating mesoderm and/or endothelial colony forming cell-like cells having in vivo blood vessel forming capacity

ABSTRACT

The present disclosure relates generally to methods and compositions useful in cell and tissue biology and therapeutics. In particular, an in vitro method for differentiating pluripotent stem cells into KDR+NCAM+APLNR+ mesoderm cells and/or SSEA5−KDR+NCAM+APLNR+ mesoderm cells is provided. The disclosed mesoderm cells may be used to generate blood vessels in vivo and/or further differentiated in vitro into endothelial colony forming cell-like cells (ECFC-like cells). Purified human cell populations of KDR+NCAM+APLNR+ mesoderm cells and ECFC-like cells are provided. Test agent screening and therapeutic methods for using the cell populations of the present disclosure are provided.

CROSS REFERENCE TO PRIOR APPLICATIONS

This application claims the benefit of U.S. Provisional patentapplication Ser. No. 62/372,907, filed Aug. 10, 2016, which isincorporated herein by reference as if set forth in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to the fields of cell and tissue biology.More particularly, the present disclosure relates to lineage-specificdifferentiation of pluripotent stem cells into mesoderm cells and/orendothelial colony forming cell-like cells (ECFC-like cells) that canform blood vessels in vivo.

BACKGROUND OF THE DISCLOSURE

Endothelial colony forming cells (ECFCs) are rare circulatingendothelial cells, particularly abundant in umbilical cord blood, withclonal proliferative potential and intrinsic in vivo vessel formingability (Yoder, M. C. et al. Blood 109, 1801-1809 (2007); Ingram, D. A.et al. Blood 105, 2783-2786 (2005); Ingram, D. A. et al. Blood 104,2752-2760 (2004); Critser, P. J. et al. Microvasc Res 80, 23-30 (2010);Au, P. et al. Blood 111, 1302-1305 (2008); Melero-Martin, J. M. et al.Circ Res 103, 194-202 (2008)). It is not understood what type of cellwithin umbilical cord blood or donor marrow gives rise to ECFCs. Whencultured primary ECFCs are injected intravenously into rodent vascularinjury models, they are recruited the site of vascular injury or tissueischemia to orchestrate initiation of a vasculogenic response (Moubarik,C. et al. Stem Cell Rev 7, 208-220 (2011); Schwarz, T. M. et al.Arterioscler Thromb Vasc Biol 32, e13-21 (2012); Saif, J. et al.Arterioscler Thromb Vasc Biol 30, 1897-1904 (2010). Human ECFCs havebeen reported to enhance vascular repair and improve blood flowfollowing myocardial infarction (Dubois, C. et al. J Am Coll Cardiol 55,2232-2243 (2010); Schuh, A. et al. Basic Res Cardiol 103, 69-77 (2008),stroke (Moubarik, C. et al. 2011), ischemic retinopathy (Stitt, A. W. etal. Prog Retin Eye Res 30, 149-166 (2011); Medina, R. J. et al. InvestOphthalmol Vis Sci 51, 5906-5913 (2010), ischemic limb injury (Schwartzet al. 2012; Saif et al. 2010; Bouvard, C. et al. Arterioscler ThrombVasc Biol 30, 1569-1575 (2010); Lee, J. H. et al. Arterioscler ThrombVasc Biol (2013), and to engraft and re-endothelialize denuded vascularsegments or implanted grafts; (Stroncek, J. D. et al. Acta Biomater 8,201-208 (2012). In subjects with peripheral arterial disease (PAD) andcritical limb ischemia (CLI), circulating or resident ECFCs may becomeprone to replicative senescence (i.e., ECFCs may lack proliferativepotential), thus rendering them impotent for autologous vascular repair.At least for these reasons, it is desirable to find alternate sources ofECFCs or other cell types that may be used for vascular repair.

Human pluripotent stem cells (hPSCs) display virtually unlimitedself-renewal capacity and ability to differentiate into any cell type inthe animal body (Robbins, R. D. et al. Curr Opin Organ Transplant 15,61-67 (2010); Broxmeyer, H. E. et al. Blood 117, 4773-4777 (2011); Lee,M. R. et al. Stem Cells 31, 666-681 (2013). The present inventors havepreviously determined a method for in vitro derivation of ECFC-likecells from hPSCs, in which the ECFC-like cells can form blood vessels invivo (PCT/US2015/020008).

It is desirable to mitigate and/or obviate one or more of the abovedeficiencies.

SUMMARY OF THE DISCLOSURE

The present disclosure is broadly summarized as relating to methods forgenerating lineage-specific mesoderm cells and/or endothelial colonyforming cell-like cells (ECFC-like cells) from hPSCs. Protocols forreproducibly differentiating hPSCs into populations of lineage-specificmesoderm and/or ECFC-like cells having in vivo blood vessel formationcapacity are provided

In an aspect, the present disclosure provides a method for generating anisolated population of human KDR⁺NCAM⁺APLNR⁺ mesoderm cells from humanpluripotent stem cells. The method comprises providing pluripotent stemcells (PSCs); inducing the pluripotent stem cells to undergo mesodermaldifferentiation, wherein the mesodermal induction comprises: i)culturing the pluripotent stem cells for about 24 hours in a mesodermdifferentiation medium comprising Activin A, BMP-4, VEGF and FGF-2; andii) replacing the medium of step i) with a mesoderm differentiationmedium comprising BMP-4, VEGF and FGF-2 about every 24-48 hoursthereafter for about 72 hours; and isolating from the cells induced toundergo differentiation the mesoderm cells, wherein the isolation ofmesoderm cells comprises: iii) sorting the mesoderm cells to select forKDR⁺NCAM⁺APLNR⁺ cells.

In an embodiment, the sorting further comprises selection ofSSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells.

In an embodiment, the mesodermal induction further comprises contactingthe cells undergoing mesodermal induction with Fc-NRP-1. In anembodiment, the mesoderm differentiation medium of step ii).

In an embodiment, the mesodermal induction further comprises contactingthe cells undergoing mesodermal induction with one or more miRNAinhibitor, wherein the one or more miRNA inhibitor inhibits an miRNAthat exhibits decreased expression in SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesodermcells relative to PSCs. In an embodiment, the miRNA inhibitor inhibitsan miRNA selected from the group consisting of: miR-221-3p, miR-1271-5p,miR-559, miR543, miR-361-3p, miR-30d-5p, miR-124-3p and miR-185-5p. Inan embodiment, the cells undergoing mesodermal induction are contactingwith one or more of an miRNA inhibitor of miR-221-3p, miR-1271-5p andmiR-559, preferably miR-221-3p.

In an embodiment, the mesodermal induction further comprises contactingthe cells undergoing mesodermal induction with one or more miRNA mimic,wherein the one or more miRNA mimic mimics an miRNA that exhibitsincreased expression in SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesoderm cells relative toPSCs. In an embodiment, the miRNA mimic mimics an miRNA selected fromthe group consisting of: miR-330-5p, miR-145-5p, miT-214-3p andmiR-497-5p. In an embodiment, the cells undergoing mesodermal inductionare cultured with one or more of an miRNA mimic of miR-330-5p,miR-145-5p and miT-214-3p, preferably miR-330-5p. In an embodiment, themesodermal induction further comprises contacting the cells undergoingmesodermal induction with a miR-214 mimic.

In an embodiment, the isolated mesoderm cells have a capacity to formblood vessels when implanted into a mammal.

In an embodiment, the method further comprises: inducing the isolatedmesoderm cells to undergo endothelial differentiation, wherein theendothelial induction comprises: culturing the isolated mesoderm cellsin an endothelial differentiation medium comprising BMP-4, VEGF andFGF-2 for about 6-8 days; and isolating from the cells induced toundergo endothelial differentiation endothelial colony forming-like(ECFC-like) cells, wherein the ECFC-like cells are CD31+ NRP-1+ andexhibit a cobblestone morphology.

In an embodiment, the isolated ECFC-like cells are further characterizedby one or more of CD144+, KDR+ and α-SMA-expression.

In an embodiment, the endothelial inducing step is carried out in theabsence of one or more of: co-culture cells, embryoid body formation andexogenous TGF-β inhibition.

In an embodiment, the isolated ECFC-like cells have a capacity to formblood vessels when implanted into a mammal in the absence ofco-implanted cells.

In an aspect, an isolated population of human KDR⁺NCAM⁺APLNR⁺ mesodermcells is provided. The isolated KDR⁺NCAM⁺APLNR⁺ mesoderm cells have acapacity to form blood vessels when implanted into a mammal and whereinthe isolated KDR⁺NCAM⁺APLNR⁺ mesoderm cells were derived in vitro fromhuman pluripotent stem cells.

In an embodiment, the KDR⁺NCAM⁺APLNR⁺ mesoderm cells are SSEA5−. In anembodiment, the KDR⁺NCAM⁺APLNR⁺ mesoderm cells exhibit increasedexpression of one or more lateral plate-extra-embryonic mesoderm markersrelative to PSCs, wherein the lateral plate-extra-embryonic mesodermmarkers are selected from BMP4, WNT5A, NKX2-5 and HAND1. In anembodiment, the KDR⁺NCAM⁺APLNR⁺ mesoderm cells lack increased expressionof one or more axial mesoderm markers, paraxial mesoderm markers and/orintermediate mesoderm markers, relative to PSCs, wherein the axialmesoderm markers are selected from CHIRD and SHH, the paraxial mesodermmarkers are selected from PAX1, MEOX1, and TCF15, and the intermediatemesoderm markers are selected from GOSR1, PAX2 and PAX8.

In an aspect, an isolated population of human KDR⁺NCAM⁺APLNR⁺ mesodermcells obtained according to the method provided herein is provided.

In an aspect, an isolated population of human NRP-1+CD31+ endothelialcolony forming cell-like cells (ECFC-like cells) obtained according tothe method provided herein is provided.

In an aspect, a pharmaceutical composition comprising theKDR⁺NCAM⁺APLNR⁺ mesoderm cells provided herein is provided.

In an aspect, a pharmaceutical composition comprising the endothelialcolony forming cell-like cells (ECFC-like cells) provided herein isprovided.

In another aspect of the present disclosure, there is provided a methodfor transplantation in a subject in need thereof, the method comprisingproviding to the subject an isolated population of cells as describedherein.

In another aspect of the present disclosure, there is provided a methodof treating a subject in need of epithelial repair, the methodcomprising providing to the subject a therapeutically effective amountof a population of cells as described herein.

In another aspect of the present disclosure, there is provided apharmaceutical composition comprising mesoderm and/or ECFC-like cellsobtained by a method as described herein.

In another aspect of the present disclosure, there is provided a methodof examining a test agent for its ability to modify cellular activity,the method comprising:

-   -   exposing at least one of the cells of the population of cells as        described herein to a test agent and;    -   observing the effect of the test agent on one or more of cell        growth and cell viability.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing in color.Copies of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

The features of the disclosure will become more apparent in thefollowing detailed description in which reference is made to theappended drawings wherein:

FIGS. 1A-1G illustrate that NCAM and APLNR co-expressing cells withinday 4 (D4) KDR⁺ mesoderm cells (KNA⁺ mesoderm) give rise to NRP-1⁺CD31⁺endothelial cells with ECFC competence.

FIG. 1A depicts a schematic of a one-step, 2D, serum and feeder-freemesoderm lineage differentiation protocol.

FIG. 1B depicts a sorting strategy for day 4 differentiated hiPSCs.

FIG. 1C depicts a strategy for sorting KDR⁺NCAM⁺APLNR⁺ (K⁺N⁺A⁺),KDR⁺NCAM⁺APLNR⁻ (K⁺N⁺A⁻), and KDR⁺NCAM⁻APLNR⁻ (K⁺N⁻A⁻) cells and furtherdifferentiating and examining the emergence of NRP-1⁺CD31⁺ ECFC-likecells.

FIG. 1D depicts examination of sorted K⁺N⁺A⁺, K⁺N⁺A⁻ and K⁺N⁻A⁻ mesodermsub-sets that were further differentiated into an ECFC-like lineage foranother 9 days (3 plus 9 days, total of 12 days) for the emergence ofNRP-1⁺CD31⁺ cells at 8, 10 and 12 days of differentiation (threeleft-most panels); morphology of day 12 monolayers is shown (fourthpanel from left), α-SMA expression is shown (fifth panel from left); andexpression of CD31 and CD144 endothelial markers is shown (right-mostpanels).

FIG. 1E depicts a bar graph showing that hiPSC-ECFC-like cells, K⁺N⁺A⁺-,K⁺N⁺A⁻-, and K⁺N⁻A⁻-mesoderm-derived NRP-1⁺CD31⁺ cells exhibiteddiffering levels of clonal proliferative potential.

FIG. 1F depicts images of various qualities of in vivo human bloodvessel formation by hiPSC-ECFC-like cells, K⁺N⁺A⁺-, K⁺N⁺A⁻-, andK⁺N⁺A⁻-mesoderm-derived NRP-1⁺CD31⁺.

FIG. 1G depicts a bar graph showing that hiPSC-ECFC-like cells,K⁺N⁺A⁺-ECFC-like cells, K⁺N⁺A⁻-ECs, and K⁺N⁺A⁻-ECs form various amountsof functional hCD31⁺ vessels.

FIGS. 2A-2C illustrate that human iPS cells differentiated for 3-5 daysusing the protocol provided herein generate cells expressing mesodermmarkers and lacking typical endothelial surface marker expression.

FIG. 2A illustrates that APLNR⁺ cells were found in the KDR⁺NCAM⁺mesoderm sub-set but not in the KDR⁻NCAM⁻ sub-set.

FIG. 2B depicts levels of KDR⁺ cells at days 3, 4, and 5 ofdifferentiation.

FIG. 2C depicts levels of endothelial marker (CD31, NRP-1 and CD144)expression in day 4-differentiated cells.

FIGS. 3A-3B illustrate that direct in vivo differentiation of D4 KNA⁺mesoderm cells isolated without SSEA5 depletion formed robust humanblood vessels and produced endoderm-derived cell-like derivatives.

FIG. 3A depicts a sorting strategy for day 4-differentiated hiPSCs.

FIG. 3B shows that sorted APLNR⁻ mesoderm cells produced a teratomaafter 2 months of in vivo implantation (left panel) and APLNR⁺ mesodermcells formed robust in vivo human blood vessels filled with host murinered blood cells (right panel; blue open arrows) with accompanyingendoderm-derived cell like derivatives (right panel; pink closedarrows).

FIGS. 4A-4D illustrate direct in vivo differentiation of D4 SSEA5depleted KNA⁺ mesoderm cells formed robust human blood vessels withoutgiving rise to teratoma or endoderm-derived cell-like derivatives.

FIG. 4A depicts a sorting strategy for day 4 differentiated hiPSCs.

FIG. 4B depicts formation of robust functional in vivo vessels bySSEA5⁻KNA⁺ cells (blue arrows, left panel) and the absence of robustfunctional in vivo vessels by SSEA5⁻KNA⁻ cells (white arrows, rightpanel).

FIG. 4C depicts a bar graph illustrating in vivo formation of functionalblood vessels by SSEA5⁻KNA⁺ cells and SSEA5⁻KNA⁻ cells.

FIG. 4D depicts images of NRP-1⁺CD31⁺ blood vessels formed in vivo 8days after implantation of SSEA5⁻KNA⁺ cells.

FIGS. 5A-5C illustrate that D4 SSEA5-depleted KNA⁺ mesoderm cellsdisplay transcripts typically enriched in lateral plate/extra-embryonicmesoderm cells, and exhibit enhanced formation of NRP-1⁺CD31⁺ cells withECFC competence upon in vitro ECFC differentiation.

FIG. 5A depicts gene expression analysis of lateralplate-extra-embryonic mesoderm markers, axial, paraxial and intermediatemesoderm markers in SSEA5⁻KNA⁺ cells and hiPSCs.

FIG. 5B depicts sorted SSEA5⁻KNA⁺ cells that were further differentiatedinto ECFC lineage for another 8 days (4 plus 8, total of 12 days); atday 12, SSEA5⁻KNA⁺ cells produced ≥3 fold more NRP-1⁺CD31⁺ cellscompared to NRP-1⁺CD31⁺ cells derived from hiPSCs (without a day 4sorting step) (left bar graph); morphology of a monolayer of NRP1⁺CD31⁺cells derived from SSEA5⁻KNA⁺ mesoderm cells, lack of α-SMA expression(middle panels); and uniform co-expression of CD31 and CD144 endothelialmarkers (right panel) in the NRP1⁺CD31⁺ cells derived from SSEA5⁻KNA⁺mesoderm cells are also shown.

FIG. 5C depicts a bar chart showing clonal proliferative potential ofhiPSC-ECFC-like cells, SSEA5⁻KNA⁺ mesoderm-derived NRP-1⁺CD31⁺ cells andSSEA5⁻KNA⁻ mesoderm-derived NRP-1⁺CD31⁺ cells.

FIGS. 6A-6G illustrate that Fc-NRP-1 mediates VEGF-KDR signaling throughp130^(Cas)/Pyk2 activation and enhances formation of SSEA5⁻KNA⁺ mesodermcells from hiPSCs.

FIG. 6A depicts a schematic representation of NRP-1, KDR, Fc-NRP-1,NRP-1-B and VEGF₁₆₅ functions in endothelial cells.

FIG. 6B depicts a Western blot showing KDR phosphorylation; KDRphosphorylation was observed in VEGF stimulated groups and Fc-NRP-1dimer treatment increased phosphorylation of KDR compared to controltreated cells; decreased phosphorylation was observed in NRP-1-B treatedcells.

FIG. 6C depicts a Western blot showing p130^(Cas)/Pyk2 phosphorylation;increased p130^(Cas)/Pyk2 phosphorylation was observed in Fc-NRP-1 dimertreated cells compared to NRP-1-B treated cells.

FIG. 6D depicts gene expression analysis for various VEGF-A isoformsbetween hiPSCs and SSEA5⁻KNA⁺ mesoderm cells.

FIG. 6E depicts a bar chart showing VEGF production from hiPSCs andSSEA5⁻KNA⁺ mesoderm cells at day 2 and day 4 of differentiation in thepresence or absence of added VEGF₁₆₅ in the culture media.

FIG. 6F depicts a schematic showing a mesoderm lineage differentiationprotocol in the presence of Fc-NRP-1 dimer.

FIG. 6G depicts a bar graph showing that dimeric Fc-NRP-1 treatment ofdifferentiating hiPSC caused more than a 2-fold increase in productionof SSEA5⁻KNA⁺ mesoderm cells compared to Fc-NRP-1 untreated cells.

FIGS. 7A-7E illustrate that specific miRNA mimics and inhibitors enhanceSSEA5⁻KNA⁺ mesoderm generation from hiPSCs.

FIG. 7A depicts fold change in expression of various miRNAs in Day 4SSEA5⁻KNA⁺ mesoderm cells relative to hiPSCs (indicated by 0 on Y-axis).

FIG. 7B depicts a schematic showing a mesoderm lineage differentiationprotocol that involves contacting the cells undergoing mesoderminduction with specific miRNA mimics or inhibitors or both mimics andinhibitors (3m3i) or mimic/inhibitor controls.

FIG. 7C depicts a bar chart showing frequency of Day 4 SSEA5⁻KNA⁺mesoderm cells obtained using the protocols of FIG. 7B.

FIG. 7D depicts a bar chart showing the relative expression (y-axis) ofvarious miRNAs (x-axis) in Day 4 SSEA5⁻KNA⁺ mesoderm cells obtainedusing the protocols of FIG. 7B.

FIG. 7E depicts a bar chart showing the relative expression (y-axis) ofvarious miRNAs (x-axis) in Day 4 SSEA5⁻KNA⁺ mesoderm cells obtainedusing the protocols of FIG. 7B.

FIGS. 8A-8D illustrate that miR-214-3p targets CLDN6 in differentiatinghiPSC and enhances formation of SSEA5⁻KNA⁺ mesoderm cells.

FIG. 8A depicts bar charts showing relative expression levels (y-axis)of miR-214 (left) and its putative target CLDN6 (right) in hiPSCs andSSEA5⁻KNA⁺ mesoderm cells.

FIG. 8B depicts a bar chart showing production of SSEA5⁻KNA⁺ mesodermcells and a GFP reporter control vector (y-axis) when miR-214 wasover-expressed in cells undergoing mesodermal induction.

FIG. 8C depicts results of a luciferase reporter assay confirming thatmiR-214 directly regulates expression of wild type (WT) CLND6.

FIG. 8D depicts results of a differential gene expression analysisbetween hiPSC and SSEA5⁻KNA⁺ mesoderm cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure generally relates to methods for in vitrodifferentiation of pluripotent cells, such as, for example, humanembryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs)(collectively, human pluripotent stem cells (hPSCs)), intolineage-specific mesoderm cells and, optionally, further differentiatingthe lineage-specific mesoderm cells into endothelial colony formingcell-like cells (ECFC-like cells). Surprisingly, the inventors havefound that the mesoderm cells generated and isolated using the methodprovided herein can generate blood vessels in vivo. In variousembodiments of the method provided herein, the resulting ECFC-like cellsmay be further grown into blood vessels in vivo in the absence ofco-culture and/or co-implantation cells.

I: Definitions

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this disclosure belongs.

As used herein, “pluripotent cell” refers to a cell that has thepotential to differentiate into any cell type, for example, cells of anyone of the three germ layers: endoderm, mesoderm, or ectoderm.

As used herein, “embryonic stem cells”, “ES cells” or “ESCs” refer topluripotent stem cells derived from early embryos.

As used herein, “induced pluripotent stem cells,” “iPS cells” or “iPSCs”refer to a type of pluripotent stem cell that has been prepared from anon-pluripotent cell, such as, for example, an adult somatic cell, or aterminally differentiated cell, such as, for example, a fibroblast, ahematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like,by introducing into the non-pluripotent cell or contacting thenon-pluripotent cell with one or more reprogramming factors.

As used herein, “mesodermal differentiation medium” refers to anynutrient medium that supports and/or enhances differentiation ofpluripotent cells into cells of the mesoderm lineage.

As used herein, “mesoderm” refers to the middle of three primary germlayers in an early embryo (the other two layers being ectoderm andendoderm). There are four components or classes of mesoderm, includingaxial mesoderm, paraxial mesoderm, intermediate mesoderm and lateralplate/extra-embryonic mesoderm. Mesoderm comprises “mesoderm cells”,also referred to as “mesodermal cells”.

As used herein, miRNA mimic” refers to double-stranded RNAoligonucleotides designed to “mimic” native/endogenous miRNA activity.miRNA mimics supplement endogenous microRNA activity to discover thefunctional roles of individual microRNAs.

As used herein, miRNA inhibitor” refers to single-stranded RNAoligonucleotides designed to “inhibit” native/endogenous miRNA activity.miRNA inhibitors suppress the function of endogenous miRNAs, increasethe expression of the target gene, and attenuate the presentation of thephenotype.

As used herein, “endothelial differentiation medium” refers to anynutrient medium that supports and/or enhances differentiation ofpluripotent cells into cells of the endothelial lineage.

As used herein, “endothelial growth medium” refers to any medium that issuitable for maintaining cells of the endothelial lineage.

As used herein, “endothelial colony forming cell” and “ECFC” refer toprimary endothelial cells found in the blood that display the potentialto proliferate and form an endothelial colony from a single cell andhave a capacity to form blood vessels in vivo in the absence ofco-implanted or co-cultured cells.

As used herein, “cord blood ECFC” and “CB-ECFC” refer to primary ECFCsthat are derived from umbilical cord blood.

As used herein, “endothelial colony forming cell-like cell” and“ECFC-like cell” refer to non-primary endothelial cells that aregenerated in vitro from human pluripotent stem cells (hPSCs). ECFC-likecells have various characteristics of ECFCs, at least including thepotential to proliferate and form an endothelial colony from a singlecell and have a capacity to form blood vessels in vivo in the absence ofco-implanted or co-cultured cells.

As used herein, the terms “proliferation potential” and “proliferativepotential” refer to the capacity of a cell to divide when providedappropriate growth promoting signals.

As used herein, the terms “high proliferation potential”, “highproliferative potential” and “HPP” refer to the capacity of a singlecell to divide into more than about 2000 cells in a 14 day cell culture.Preferably, HPP cells have a capacity to self-replenish. For example,the HPP-ECFC-like cells provided herein have a capacity toself-replenish, meaning that an HPP-ECFC-like cell can give rise to oneor more HPP-ECFC-like cells within a secondary HPP-ECFC-like colony whenreplated in vitro. In some embodiments, HPP-ECFC-like cells may alsohave the ability to give rise to one or more of LPP-ECFC-like cells andECFC-like cell clusters within a secondary HPP-ECFC-like colony whenreplated in vitro.

As used herein, the terms “low proliferation potential” “lowproliferative potential” and “LPP” refer to the capacity of a singlecell to divide into about 51-2000 cells in a 14 day cell culture. Insome embodiments, LPP-ECFC-like cells may also have the ability to giverise to ECFC-like cell clusters. However, LPP-ECFC-like cells do nothave a capacity to give rise to secondary LPP-ECFC-like cells orHPP-ECFC-like cells.

II: Methods of Differentiating Pluripotent Cells into Mesoderm Cells

In an aspect, the method provided herein involves at least three steps:

A. providing pluripotent stem cells (PSCs);

B. inducing the pluripotent stem cells to undergo mesodermaldifferentiation, wherein the mesodermal induction comprises: i)culturing the pluripotent stem cells for about 24 hours in a mesodermdifferentiation medium comprising Activin A, BMP-4, VEGF and FGF-2; andii) replacing the medium of step i) with a mesoderm differentiationmedium comprising BMP-4, VEGF and FGF-2 about every 24-48 hoursthereafter for about 72 hours; and

C. isolating from the cells induced to undergo differentiation themesoderm cells, wherein the isolation of mesoderm cells comprises: i)sorting the mesoderm cells to select for KDR⁺NCAM⁺APLNR⁺ cells.

In various embodiments, the method includes one or more of the followingfurther steps:

D. sorting the mesoderm cells to select for SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells;

E. culturing the cells undergoing mesodermal induction with Fc-NRP-1;

F. culturing the cells undergoing mesodermal induction with one or moremiRNA inhibitor, one or more miRNA mimic, or a combination thereof;

G. inducing differentiation of the isolated mesoderm cells into cells ofthe endothelial lineage; and

H. isolating ECFC-like cells from the differentiated cells of theendothelial lineage.

Each step in the aforementioned method is described further hereinbelow.

A. Pluripotent Stem Cell Culture

In one aspect, a method for generating an isolated population ofmesoderm and/or ECFC-like cells in vitro from pluripotent cells isprovided. Pluripotent cells that are suitable for use in the methods ofthe present disclosure can be obtained from a variety of sources. Forexample, one type of suitable pluripotent cell is an embryonic stem (ES)cell derived from the inner cell mass of a blastocyst. Methods forobtaining various types of ES cells, such as mouse, rhesus monkey,common marmoset, and human, are well known. The source of ES cells usedin the method may be, for example, one or more established ES celllines. Various ES cell lines are known and the conditions for theirgrowth and propagation have been defined. It is contemplated herein thatvirtually any ES cell or ES cell line may be used with the methodsdisclosed herein. In one embodiment, the pluripotent cell is an inducedpluripotent stem (iPS) cell derived by reprogramming somatic cells.Induced pluripotent stem cells have been obtained by various knownmethods. It is contemplated herein that virtually any iPS cell or cellline may be used with the methods disclosed herein. In otherembodiments, the pluripotent cell is an embryonic stem cell derived bysomatic cell nuclear transfer, in which a donor nucleus is transferredinto a spindle-free oocyte. Various methods for producing stem cells bynuclear transfer are known. It is contemplated herein that virtually anyES cells or cell line derived by somatic cell nuclear transfer may beused with the methods disclosed herein.

In one embodiment, pluripotent cells are cultured under conditionssuitable for maintaining pluripotent cells in an undifferentiated state.Methods for maintaining pluripotent cells in vitro, i.e., in anundifferentiated state, are well known. In one embodiment, pluripotentcells are cultured for about two days under conditions suitable formaintaining pluripotent cells in an undifferentiated state. For example,in the Examples below, hES and hiPS cells were maintained in mTeSR1complete medium on Matrigel™ in 10 cm² tissue culture dishes at 37° C.and 5% CO₂ for about two days.

Additional and/or alternative methods for culturing and/or maintainingpluripotent cells may be used. For example, as the basal culture medium,any of TeSR, mTeSR1 alpha.MEM, BME, BGJb, CMRL 1066, DMEM, Eagle MEM,Fischer's media, Glasgow MEM, Ham, IMDM, Improved MEM Zinc Option,Medium 199 and RPMI 1640, or combinations thereof, may be used forculturing and or maintaining pluripotent cells.

The pluripotent cell culture medium used may contain serum or it may beserum-free. Serum-free refers to a medium comprising no unprocessed orunpurified serum. Serum-free media can include purified blood-derivedcomponents or animal tissue-derived components, such as, for example,growth factors. The pluripotent cell medium used may contain one or morealternatives to serum, such as, for example, knockout Serum Replacement(KSR), chemically-defined lipid concentrated (Gibco) or glutamax(Gibco).

Methods for splitting or passaging pluripotent cells are well known. Forexample, in the Examples below, after pluripotent cells were plated,medium was changed on days 2, 3, and 4 and cells were passaged on day 5.Generally, once a culture container is full (i.e., 70-100% confluence),the cell mass in the container is split into aggregated cells or singlecells by any method suitable for dissociation and the aggregated orsingle cells are transferred into new culture containers for passaging.Cell “passaging” or “splitting” is a well-known technique for keepingcells alive and growing cells in vitro for extended periods of time.

B. Directed Differentiation of Pluripotent Cells into Mesoderm Cells.

In one aspect of the method disclosed, in vitro pluripotent cells areinduced to undergo mesodermal differentiation, also referred to as astep of “mesodermal induction”. Various methods, including cultureconditions, for inducing differentiation of pluripotent cells into cellsof the mesodermal lineage are known in the art. In the protocol providedherein it is preferable to induce differentiation of pluripotent cellsin a chemically defined medium. For example, Stemline II serum-freehematopoietic expansion medium can be used as a basal mesodermaldifferentiation medium. In the protocol provided herein various growthfactors are used to promote differentiation of pluripotent cells intocells of the mesodermal lineage. For example, Activin A, vascularendothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2)and bone morphogenetic protein 4 (BMP-4) are included in a chemicallydefined differentiation medium to induce differentiation of pluripotentcells into cells of the mesodermal lineage.

In one embodiment of the protocol provided herein, after 2 days (−D2) ofculture in a basal culture medium (e.g., mTeSR1), differentiation ofpluripotent cells was directed toward the mesodermal lineage bycontacting the cells for 24 hours with an endothelial differentiationmedium comprising an effective amount of Activin A, BMP-4, VEGF andFGF-2. Following 24 hours of differentiation, Activin A was removed fromthe culture by replacing the mesodermal differentiation medium with anmesodermal differentiation medium comprising an effective amount ofBMP-4, VEGF and FGF-2. By “effective amount”, we mean an amounteffective to promote differentiation of pluripotent cells into cells ofthe mesodermal lineage. Further replacement of the mesodermaldifferentiation medium comprising an effective amount of BMP-4, VEGF andFGF-2 may be done every 1-2 days for about 3 days (i.e., to D4).

Activin A is a member of the TGF-B superfamily that is known to activatecell differentiation via multiple pathways. Activin-A facilitatesactivation of mesodermal specification but is not critical forendothelial specification and subsequent endothelial amplification. Inone embodiment, the mesodermal differentiation medium comprises ActivinA in a concentration of about 5-25 ng/mL. In one preferred embodiment,the mesodermal differentiation medium comprises Activin A in aconcentration of about 10 ng/mL.

Bone morphogenetic protein-4 (BMP-4) is a ventral mesoderm inducer thatis expressed in adult human bone marrow (BM) and is involved inmodulating proliferative and differentiative potential of hematopoieticprogenitor cells. Additionally, BMP-4 can modulate early hematopoieticcell development in human fetal, neonatal, and adult hematopoieticprogenitor cells. In one embodiment, the mesodermal differentiationmedium comprises BMP-4 in a concentration of about 5-25 ng/mL. In onepreferred embodiment, the mesodermal differentiation medium comprisesBMP-4 in a concentration of about 10 ng/mL.

Vascular endothelial growth factor (VEGF) is a signaling proteininvolved in embryonic circulatory system formation and angiogenesis. Invitro, VEGF can stimulate endothelial cell mitogenesis and cellmigration. In one embodiment, the mesodermal differentiation mediumcomprises VEGF in a concentration of about 5-50 ng/mL. In one preferredembodiment, the mesodermal differentiation medium comprises VEGF in aconcentration of about 10 ng/mL. In one particularly preferredembodiment, the mesodermal differentiation medium comprises VEGF₁₆₅ in aconcentration of about 10 ng/mL.

Basic fibroblast growth factor, also referred to as bFGF or FGF-2, hasbeen implicated in diverse biological processes, including limb andnervous system development, wound healing, and tumor growth. bFGF hasbeen used to support feeder-independent growth of human embryonic stemcells. In one embodiment, the mesodermal differentiation mediumcomprises FGF-2 in a concentration of about 5-25 ng/mL. In one preferredembodiment, the mesodermal differentiation medium comprises FGF-2 in aconcentration of about 10 ng/mL.

The method disclosed herein does not require co-culture with supportivecells, such as, for example, OP9 stromal cells, does not requireembryoid body (EB) formation and does not require exogenous TGF-βinhibition.

C. Isolating KDR⁺NCAM⁺APLNR⁺ (KNA⁺) Mesoderm Cells

In an aspect, KDR⁺NCAM⁺APLNR⁺ cells are selected and isolated from thepopulation of cells induced to undergo mesodermal differentiation.Methods, for selecting cells having one or more specific molecularmarkers are known in the art. For example, cells may be selected basedon expression of various transcripts by flow cytometry, includingfluorescence-activated cell sorting, or magnetic-activated cell sorting.

In one embodiment, KDR⁺NCAM⁺APLNR⁺ cells are selected from a populationof cells undergoing mesodermal differentiation, as described herein, onday 4 of differentiation. In one preferred embodiment, KDR⁺ cells areselected from a population of cells undergoing mesodermaldifferentiation and then from the selected KDR⁺ cells, NCAM*APLNR⁺ cellsare selected, thereby yielding a population of KDR⁺NCAM⁺APLNR⁺ cells.

In the Examples below, mesoderm cells were harvested after day 4 ofdifferentiation and made into a single cell suspension. Cells werecounted and prepared for antibody staining with anti-human antibodies toKDR, NCAM and APLNR. KDR⁺NCAM⁺APLNR⁺ cells were gated/selected andsorted using flow cytometry.

The mesoderm subsets identified herein display gene products consistentwith known subsets of human mesoderm. These specific mesoderm subsetshave not previously been noted to give rise to human ECFCs.

In one embodiment, the selected cells have a capacity for in vivo vesselformation. This result was unexpected. Specific types of mesoderm areexpressed in early human development, and those cells that give rise toangioblast cells (the first mesoderm-derived cells that furtherdifferentiate into endothelial cells) are predicted to be derived fromextra-embryonic/lateral plate mesoderm. Accordingly, a skilled personwould predict that mesoderm cells would differentiate at specific timesin specific places to form the first blood vessels via vasculogenesis. Askilled person would not predict that mesoderm cells would display theability to give rise to ECFCs and form human blood vessels in an adultimmunodeficient mouse, at least because there are no mesoderm cells thatexist in adult mice (they have already committed to lineage fates duringembryogenesis and are now only represented by their specified lineageprogeny).

D. Selection of SSEA5⁻KDR⁺NCAM⁺APLNR⁺ Cells

In one embodiment, SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells are selected from apopulation of cells undergoing mesodermal differentiation, as describedherein, on day 4 of differentiation. In one preferred embodiment,SSEA5⁻KDR⁺ cells are selected from a population of cells undergoingmesodermal differentiation and then from the selected SSEA5⁻KDR⁺ cells,NCAM⁺APLNR⁺ cells are selected, thereby yielding a population ofSSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells.

The inventors found that negative selection of Day 4 differentiatedcells with an SSEA5 antibody allowed identification and subsequentremoval of an undifferentiated or partially differentiated hiPSCsmixture from day 4 differentiated cells. This was achieved by performingin vivo implantation of SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells and KDR⁺NCAM⁺APLNR⁺cells (without negative selection for SSEA5) in the same animal. Oneabdominal side received SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells and other abdominalside of the same animal received KDR⁺NCAM⁺APLNR⁺ cells. ImplantedKDR⁺NCAM⁺APLNR⁺ cells formed blood vessels and endoderm derivatives,whereas implanted SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells formed blood vessels, anddid not form endoderm and/or teratomas. Accordingly, one advantage ofthe SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesoderm cells provided herein is that theycan be used to achieve selective ECFC blood vessel formation in vivowithout formation of endodermal derivatives.

E. Mesodermal Induction with Fc-NRP-1

The inventors contemplated that stimulating KDR signalling in the cellsundergoing mesoderm induction might increase mesoderm production. In oneembodiment of the protocol provided herein, following 24 hours ofdifferentiation in Activin-A containing medium, the mesodermaldifferentiation medium was replaced with a mesodermal differentiationmedium comprising an effective amount of Fc-NRP-1, BMP-4, VEGF andFGF-2. By “effective amount”, we mean an amount effective to promotedifferentiation of pluripotent cells into cells of the mesodermallineage. Further replacement of the mesodermal differentiation mediumcomprising an effective amount of Fc-NRP-1, BMP-4, VEGF and FGF-2 may bedone, for example, every 1-2 days for about 3 days (i.e., to D4). Theinventors found that addition of Fc-NRP-1 to the mesoderm inductionprotocol improved mesoderm generation. This result is surprising, atleast because NRP-1 (the Fc-NRP-1 acts as a surrogate for NRP-1) andVEGF₁₆₅ (the ligand that binds to Fc-NRP1 or endogenous NRP-1), are notexpressed in the cells undergoing mesoderm induction. The othermolecules that are known to activate KDR signaling are VEGF₁₆₅ andNRP-1. However, the inventors' studies show that neither of thesemolecules are endogenously expressed in the cells undergoing day 4mesoderm differentiation. The inventors' identification of a solublemolecule (which could be used to supplement culture media) that canactivate KDR signaling, thereby enhancing mesoderm formation, isadvantageous for augmenting production of ECFC mesoderm cells.

F. Mesodermal Induction with miRNA Inhibitors and/or Mimics.

In one embodiment of the protocol provided herein, cells being inducedto undergo mesodermal differentiation are exposed to one or more miRNAinhibitors, mimics, or a combination thereof. The inventors haveidentified a set of miRNAs that are downregulated inSSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells (miR-221-3p, miR-1271-5p, miR-559, miR-543,miR-361-3p, miR-30d-5p, miR-124-3p and miR-185-5p) and a set of miRNAsthat were identified as being upregulated in SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells(miR-330-5p, miR-145-5p, miR-214-3p and miR-497-5p). The inventors havefound that transfecting the cells undergoing mesoderm induction with oneor more agents that mimic specific miRNAs that are upregulated inSSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells increases the frequency ofSSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells generated from PSCs. The inventors havefound that transfecting the cells undergoing mesoderm induction with oneor more agents that inhibit specific miRNAs that were identified asbeing downregulated in SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells increases thefrequency of SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cells generated from PSCs. Theinventors have also found that transfecting the cells undergoingmesoderm induction with a combination of specific miRNA mimics andinhibitors increases the frequency of SSEA5⁻KDR⁺NCAM⁺APLNR⁺ cellsgenerated from PSCs.

In one embodiment of the protocol provided herein, after 2 days (−D2) ofculture in a basal culture medium, differentiation of pluripotent cellswas directed toward the mesodermal lineage by contacting the cells for24 hours with an endothelial differentiation medium comprising aneffective amount of Activin A, BMP-4, VEGF and FGF-2. During this first24 hours (i.e., on D0), the cells were transfected with one of 4treatments: i) 3 miRNA mimics; ii) 3 miRNA inhibitors; iii) 3 miRNAmimics and 3 miRNA inhibitors; or iv) a control. Following 24 hours ofdifferentiation, Activin A was removed from the culture by replacing themesodermal differentiation medium with an mesodermal differentiationmedium comprising an effective amount of BMP-4, VEGF and FGF-2. Thecells were transfected again, with the same treatment, on day 2 ofmesodermal induction. Further replacement of the mesodermaldifferentiation medium comprising an effective amount of BMP-4, VEGF andFGF-2 may be done every 1-2 days for about 3 days (i.e., to D4). On day4, cells were sorted and isolated, as described above.

G. Directed Differentiation of Isolated Mesoderm Cells into Cells of theEndothelial Lineage.

In one aspect of the method disclosed, the isolated mesoderm cells areinduced to undergo endothelial differentiation. Various methods,including culture conditions, for inducing differentiation of mesodermcells into cells of the endothelial lineage are known in the art. In theECFC-like cell protocol provided herein it is preferable to inducedifferentiation of endothelial cells in a chemically defined medium. Forexample, Stemline II serum-free hematopoietic expansion medium can beused as a basal endothelial differentiation medium. In the ECFC-likecell protocol provided herein various growth factors are used to promotedifferentiation of pluripotent cells into cells of the endotheliallineage, including ECFC-like cells. For example, VEGF, FGF-2 and BMP-4are included in a chemically defined differentiation medium to inducedifferentiation of isolated mesoderm cells into cells of the endotheliallineage, including ECFC-like cells.

In one embodiment of the ECFC-like cell protocol provided herein,isolated mesoderm cells are cultured with an endothelial differentiationmedium comprising an effective amount of BMP-4, VEGF and FGF-2. By“effective amount”, we mean an amount effective to promotedifferentiation of isolated mesoderm cells into cells of the endotheliallineage, including ECFC-like cells. Further replacement of theendothelial differentiation medium comprising an effective amount ofBMP-4, VEGF and FGF-2 may be done every 1-2 days.

The method disclosed herein does not require co-culture with supportivecells, such as, for example, OP9 stromal cells, does not requireembryoid body (EB) formation, and does not require exogenous TGF-βinhibition.

H. Isolating ECFC-Like Cells from the Differentiated Endothelial Cells

In one embodiment of the method disclosed herein, CD31⁺ NRP-1+ cells areselected and isolated from the population of cells undergoingendothelial differentiation. For example, in one embodiment, CD31⁺NRP-1+ cells are selected from a population of cells undergoingendothelial differentiation, as described herein, on day 10, 11 or 12 ofdifferentiation. In one preferred embodiment, CD31⁺ NRP-1+ cells areselected from the population of cells undergoing endothelialdifferentiation on day 12 of differentiation. The inventors have foundthat the day 12 population of cells undergoing endothelialdifferentiation contains a higher percentage of NRP-1+ cells relative tocell populations that are present on other days of differentiation.

In the Examples below, adherent ECs were harvested after day 12 ofdifferentiation and made into a single cell suspension. Cells werecounted and prepared for antibody staining with anti-human CD31, CD144and NRP-1. CD31⁺ CD144+NRP-1+ cells were sorted and selected using flowcytometry.

In one embodiment, the selected cells exhibit a cobblestone morphology,which is typical of ECs, including ECFCs.

In one embodiment, the selected cells have a capacity for in vivo vesselformation in the absence of co-culture and/or co-implanted cells, whichis typical of ECFCs.

III. Isolated Cell Populations Isolated Mesoderm Cell Populations

In one embodiment, an isolated population of human KDR⁺NCAM⁺APLNR⁺mesoderm cells is provided. In one embodiment, the purified human cellpopulation of KDR⁺NCAM⁺APLNR⁺ mesoderm cells provided is generated usingthe in vitro method for generating mesoderm cells from hPSCs disclosedherein. The isolated KDR⁺NCAM⁺APLNR⁺ mesoderm cells of the populationhave a capacity to give rise to ECFCs and the capacity for blood vesselformation in vivo. In one embodiment, the KDR⁺NCAM⁺APLNR⁺ mesoderm cellsof the population are further characterized by increased expression ofone or more lateral plate-extra-embryonic mesoderm markers (e.g., BMP4,WNT5A, NKX2-5 and/or HAND1) relative to PSCs. In one embodiment, theKDR⁺NCAM⁺APLNR⁺ mesoderm cells of the population are furthercharacterized by a lack of increased expression of one or more axialmesoderm markers (e.g., CHIRD and/or SHH), paraxial mesoderm markers(e.g., PAX1, MEOX1, and TCF15) and/or intermediate mesoderm markers(e.g., GOSR1, PAX2 and PAX8), relative to PSCs.

In one embodiment, an isolated population of human SSEA5⁻KDR⁺NCAM⁺APLNR⁺mesoderm cells is provided. In one embodiment, the purified human cellpopulation of SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesoderm cells provided is generatedusing the in vitro method for generating mesoderm cells from hPSCsdisclosed herein. The isolated SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesoderm cells ofthe population have a capacity for ECFC formation and blood vesselformation in vivo. In one embodiment, the SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesodermcells of the population are further characterized by increasedexpression of one or more lateral plate-extra-embryonic mesoderm markers(e.g., BMP4, WNT5A, NKX2-5 and/or HAND1) relative to PSCs. In oneembodiment, the SSEA5⁻KDR⁺NCAM⁺APLNR⁺ mesoderm cells of the populationare further characterized by a lack of increased expression of one ormore axial mesoderm markers (e.g., CHIRD and/or SHH), paraxial mesodermmarkers (e.g., PAX1, MEOX1, and TCF15) and/or intermediate mesodermmarkers (e.g., GOSR1, PAX2 and PAX8), relative to PSCs.

In one preferred embodiment, the isolated mesoderm cell population issubstantially pure. In one embodiment, 28% of total live cells at day 4of mesoderm differentiation are KDR⁺NCAM⁺APLNR⁺. In one embodiment, 18%of total live cells at day 4 of mesoderm differentiation areSSEA5⁻KDR⁺NCAM⁺APLNR⁺.

Isolated ECFC-Like Cell Populations

In one embodiment, an isolated population of human NRP-1⁺/CD31⁺ECFC-like cells is provided. In one embodiment, the purified human cellpopulation of NRP-1⁺/CD31⁺ ECFC-like cells provided is generated usingthe in vitro method for generating ECFC-like cells from hPSCs disclosedherein.

In the Examples below, the method disclosed herein is used to generate apurified human cell population of NRP-1+ and CD31⁺ ECFC-like cells, froman isolated subset of mesoderm cells. The isolated ECFC-like cells ofthe population exhibit cobblestone morphology and have a capacity forblood vessel formation in vivo without co-culture and/or co-implantedcells. In one embodiment, the ECFC-like cells of the population arefurther characterized by one or more of CD144+, KDR⁺ and α-SMA−.

In one embodiment, at least some of the ECFC-like cells in thepopulation generated from the isolated mesoderm cells have a highproliferation potential that is similar to the proliferation potentialof ECFC's generated in vitro using the inventor's previoushPSC-ECFC-lice cell protocol.

In one preferred embodiment, the isolated ECFC-like cell population issubstantially pure.

In the Examples herein, cells in the ECFC-like cell populationsgenerated from the disclosed mesoderm cells can form blood vessels whenimplanted in vivo in a mammal, even in the absence of supportive cells.

Various techniques for measuring in vivo vessel formation are known andcan be used.

The capacity to form blood vessels in vivo in the absence of exogenoussupportive cells is one indicator that the cells produced using themethods disclosed herein are ECFCs.

IV. Use of Mesoderm and/or ECFC-Like Cells Disclosed Herein

In contrast to known mesoderm cell lines and other known isolatedmesoderm cells described previously, the mesoderm cells generated usingthe method disclosed herein can be generated in vitro and used to formblood vessel tissue in vivo for various clinical applications, asdescribed below.

In contrast to ECFCs, which are primary cells, the ECFC-like cellsgenerated using the method disclosed herein can be generated in vitro ina volume that can be useful for various clinical applications, asdescribed below.

A. Therapy

In one aspect, methods, cells and compositions suitable for celltransplantation, cell replenishment, and/or cell or tissue replacementare provided herein. The method can comprise providing to a subject inneed thereof a therapeutically effective amount of mesoderm and/orECFC-like cells derived according to a method provided herein, wherebyproviding mesoderm and/or ECFC-like cells treats the subject. By“therapeutically effective amount”, we mean an amount effective to treata subject who is in need of epithelial repair. In a preferredembodiment, the epithelial repair is vascular epithelial repair Thecells and/or compositions provided herein may be administered to asubject in a manner that permits the mesoderm and/or ECFC-like cells tograft or migrate to an intended tissue site and reconstitute orregenerate the functionally deficient area, such as, for example, ablood vessel.

Subjects suitable for receiving therapy using the mesoderm and/orECFC-like cells provided herein include those having endothelialdysfunction and/or damage of various kinds. For example, subjects havingcardiovascular disease, myocardial infarction, cardiac stroke, orperipheral artery disease (PAD) can be suitable subjects for receivingtherapy using the mesoderm and/or ECFC-like cells of the presentdisclosure. Subjects having lung or kidney disease or damage can besuitable subjects for receiving therapy using the mesoderm and/orECFC-like cells of the present disclosure. In preferred embodiments, PADpatients developing critical limb ischemia (CLI) can be suitablesubjects for receiving therapy using the mesoderm and/or ECFC-like cellsof the present disclosure.

In one embodiment, the mesoderm and/or ECFC-like cells can be providedto a subject in the form of a pharmaceutical composition suitable forhuman administration. For example, the composition may comprise one ormore pharmaceutically acceptable carriers, buffers, or excipients. Thecomposition may further comprise, or be provided to the subject with,one or more ingredients that facilitate the engraftment mesoderm and/orECFC-like cells. For example, the pharmaceutical composition may alsocomprise, or be provided to a subject with, one or more growth factorsor cytokines (e.g., angiogenic cytokines) that promote survival and/orengraftment of transplanted cells, promote angiogenesis, modulate thecomposition of extracellular or interstitial matrix, and/or recruitother cell types to the site of transplantation.

In one embodiment, the pharmaceutical composition may be formulated,produced, and stored according to standard methods that provide propersterility and stability.

For example, in one embodiment, the mesoderm and/or ECFC-like cellsprovided herein may be directly injected into a tissue that is lackingin adequate blood flow (as determined by a physician). In oneembodiment, the mesoderm and/or ECFC-like cells provided herein may besuspended in a matrix comprised of collagen, fibronectin, or a syntheticmaterial and this gelatinous suspension of the mesoderm and/or ECFC-likecells may be directly injected into a tissue that is lacking in adequateblood flow. The concentration of mesoderm and/or ECFC-like cellsinjected into the tissue may vary, for example, from about 10,000 toabout 100,000 cells/microliter of delivery vehicle or matrix material.In some tissues, the cells may be delivered on a single occasion withrecovery of adequate blood flow whereas other tissues may requiremultiple injections and sequential injections over time to rescueadequate blood flow.

After administering the mesoderm and/or ECFC-like cells into thesubject, the effect of the treatment method may be evaluated, if desiredand the treatment may be repeated as needed or required. Therapyefficacy can be monitored by clinically accepted criteria known in theart, such as, for example, reduction in area occupied by scar tissue,revascularization of scar tissue, frequency and severity of angina; animprovement in developed pressure, systolic pressure, end diastolicpressure, subject mobility and/or quality of life.

ECFC cells can rescue an eye from hypoxia and neovascularization.Therefore, it is contemplated herein that the mesoderm and/or ECFC-likecells provided herein be used to treat various eye diseases in whichhypoxia and neovascularization occurs, such as, for example, retinopathyof prematurity, diabetic retinopathy, central vein occlusion, or maculardegeneration.

It is also contemplated that the mesoderm and/or ECFC-like cellsprovided herein may be used to coat at least a portion of the inside ofa vascular stent and optionally any area of a vessel that became denudedof endothelial cells during the stent placement. In this case, theintravenously injected mesoderm and/or ECFC-like cells would bind toareas of injury and re-endothelialize the vessels to prevent blood clotformation and/or restenosis of the vessel area in which the stent hasbeen placed.

It is known that placement of human veins (saphenous or umbilical) asgrafts into arteries of patients that have areas of stenosis andblockade of blood flow, have a high incidence of subsequent stenosis andblocked blood flow. This is associated with loss of the blood vesselendothelial cells early in the process of vessel remodeling in vivo. Itis contemplated herein that the mesoderm and/or ECFC-like cells providedherein can be intravenously injected into the vasculature of such apatient in order to re-endothelialize the implanted graft and topreserve the function of the vessel in the patient.

B. Test Agent Screening

The mesoderm and/or ECFC-like cells disclosed herein can be used toscreen for factors (such as solvents, small molecule drugs, peptides,oligonucleotides) or environmental conditions (such as cultureconditions or manipulation) that affect the characteristics of mesodermand/or ECFC-like cells and any tissues developed therefrom. In oneembodiment, test agents, such as, for example, pharmaceutical compounds,can be screened using the mesoderm and/or ECFC-like cells of the presentdisclosure to determine their effect on endothelial health and/orrepair. For example, screening may be done either because the compoundis designed to have a pharmacological effect on the endothelial cells,or because a compound designed to have effects elsewhere may haveunintended side effects on endothelial cells. In various embodiments,the mesoderm and/or ECFC-like cells herein are particularly useful fortest agent screening, at least because they are differentiated in vitrofrom cultured pluripotent cells. In contrast, CB-ECFCs are primary cellsobtained from patient blood and previously known isolated mesoderm cellsdo not have in vivo blood vessel-forming capacity. Various methods ofscreening test agent compounds are known in the art and can be used withthe mesoderm and/or ECFC-like cells disclosed herein.

For example, screening the activity of test agents may comprise: i)combining the mesoderm and/or ECFC-like cells disclosed herein with atest agent, either alone or in combination with other agents; ii)determining changes in the morphology, molecular phenotype, and/orfunctional activity of the mesoderm and/or ECFC-like cells that can beattributed to the test agent, relative to untreated cells or cellstreated with a control agent; and iii) correlating the effect of thetest agent with the observed change.

In one embodiment, cytotoxicity of a test agent on the mesoderm and/orECFC-like cells provided herein can be determined by the effect theagent has on one or more of mesoderm and/or ECFC-like cell viability,survival, morphology, and molecular phenotype and/or receptors.

In one embodiment, mesoderm and/or ECFC-like cell function can beassessed using a standard assay to observe phenotype or activity of themesoderm and/or ECFC-like cells. For example, one or more of molecularexpression, receptor binding, either in cell culture or in vivo, may beassessed using the mesoderm and/or ECFC-like cells disclosed herein.

C. Kits

In one embodiment, kits for use with methods and cells disclosed hereinare contemplated. In one embodiment, a kit can comprise adifferentiation and/or growth medium, as described herein, in one ormore sealed vials. In one embodiment, the kit can include one or morecells, such as pluripotent cells, mesoderm cells and/or ECFC-like cells,as disclosed herein. In one embodiment, the kit can include one or moremiRNA mimic, one or more miRNA inhibitor, or a combination thereof. Inone embodiment, the kit can include Fc-NRP-1. In one embodiment, the kitcan include instructions for generating mesoderm cells from pluripotentcells. In one embodiment, the kit can include instructions forgenerating ECFC-like cells from pluripotent cells. In one embodiment,kits can include various reagents for use with the present disclosure insuitable containers and packaging materials. In one embodiment, the kitcan include one or more controls for use with the present disclosure insuitable containers and packaging materials.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1: Materials and Methods

Culturing of hPSCs: Human Embryonic stem cell (hES) cell line H9(Thomson et al., 1998 Science; 282:1145-1147) and fibroblast-derivedhuman iPS cell line (DF19-9-11T) (Yu et al. 2009 Science 324:797-801)were purchased from WiCell Research institute (Madison, Wis.). Both hESand hiPSCs were maintained in mTeSR1 complete media (Stem CellTechnologies) on Matrigel in 10 cm² tissue culture dishes at 37° C. and5% CO₂. After the plating of cells, media was changed on days 2, 3, and4. Cells were passaged on Day 5. Media was aspirated and 4-5 mL ofdispase (2 mg/mL, Gibco) containing media was added to the plate andincubated at 37° C. for 3-5 minutes or until the edges of the colonieshad lifted from the plate. Dispase containing media was aspirated fromthe plate and cells were gently washed with DMEM-F12 (Gibco) 3 times toremove any residual amount of enzyme. Fresh media was then used tocollect colonies from the plate using a forceful wash and scraping witha 5 mL disposable pipette taking care to avoid bubbles. Collectedcolonies were then centrifuged at 300×g for 5 minutes. The supernatantwas aspirated and pellet was resuspended in mTeSR1 complete media. Priorto passaging, 10 cm² tissue culture dishes were coated with Matrigel for30 minutes. Unattached Matrigel was removed from the tissue culturedishes and 7 mL of mTeSR1 complete medium was added to dishes. Coloniesevenly distributed in mTeSR1 media were added to each plate. Cells werethen spread out within the dish using multiple side to side shakingmotions while avoiding any swirling. Cultures were checked for growthquality and morphology, and by performing teratoma formation assay aspreviously described (Broxmeyer et al., 2011 Blood; 117:4773-4777).

Directed differentiation of hPSCs into mesoderm cells: After 2 days(−D2) of culture in mTeSR1 media, cultures were directed toward themesodermal lineage with addition of activin A (10 ng/mL) in the presenceof FGF-2, VEGF₁₆₅, and BMP4 (10 ng/mL) for 24 hrs. The following day(D1), activin-A containing media was removed and replaced with 8 mL ofStemline II complete media (Sigma) containing FGF-2 (Stemgent), VEGF₁₆₅(R&D) and BMP4 (R&D). Media was replaced with 8 ml of fresh Stemline IIdifferentiation media on day 3. On day 4 the cells were collected forsorting by flow cytometry for KNA⁺ mesoderm cells or SSEA5⁻KNA⁺ mesodermcells.

Directed differentiation of KNA⁺ mesoderm cells or SSEA5⁻KNA⁺ mesodermcells into the EC lineage, including ECFC-like cells: Day 4 sortedmesoderm cells (KDR⁺NCAM⁺APLNR⁺ or SSEA5⁻KDR⁺NCAM⁺APLNR⁺) were furthercultured with 8 mL of Stemline II complete media (Sigma) containingFGF-2 (Stemgent), VEGF₁₆₅ (R&D) and BMP4 (R&D), which was replaced ondays 6, 8, 10 and 12. On day 10 and thereafter media was changed with 10mL of Stemline II differentiation media.

Flow cytometry: At day 12 after differentiation, adherent cells wereharvested using TrypleE and made into a single cell suspension in EGM-2medium. Cells were counted and aliquots of the cell suspension wereprepared for antibody staining. FcR blocking reagent (Miltyni Biotech)was added to prevent the non-specific binding of antibodies. Anti-humanCD31 (CD31-FITC, clone WM59 from BD Pharmingen), CD144 (CD144-PE, clone16B1 from ebioscience) and NRP-1 (NRP-1-APC, clone AD5-176 from MiltenyiBiotech) antibodies were used at concentrations that were titrated priorto use. Propidium Iodide (PI, Sigma) was added to the cell suspensionfor dead cell staining. Flow cytometric detection of the cell surfaceantigens and cells sorting were performed on an LSR II and FACS Aria(Becton Dickinson) respectively. Compensation was set by single positivecontrols using cord blood derived ECFCs. A gating of targeted cellpopulation was determined based on fluorescent minus one (FMO) controlsfor each fluorescent color.

Cell culture of sorted cells: CD31⁺, CD144⁺ or KDR⁺ and NRP-1⁺ sortedcells were centrifuged at 300×g for 5 minutes then resuspended in 50%EGM-2 and 50% complete Stemline II differentiation media. To generateECFCs from the sorted population, 2500 cells per well were seeded on rattail type I collagen coated 12 well plates. After 2 days, the media wasaspirated and three parts of EGM-2 and one part of differentiation mediawas added to the cultures. ECFC colonies appeared as tightly adherentcells and exhibited cobblestone morphology on day 7. On occasion,cloning cylinders were used to isolate ECFC colonies from heterogeneouscell populations. Cloning of endothelial cell clusters was performed toisolate pure populations of highly proliferative endothelial cells asdescribed previously (Yoder et al., 2007; Ingram et al., 2005).Confluent ECFCS were passage by plating 10,000 cells per cm² as aseeding density and maintain ECFCs in complete endothelial growth media(collagen coated plates and cEGM-2 media) with media change every otherday as described previously (Yoder et al., 2007; Ingram et al., 2005).

Immunochemistry: ECFCs were fixed with 4% (w/v) paraformaldehyde for 30minutes and permeabilized with 0.1% (v/v) TritonX-100 in PBS for 5minutes. After blocking with 10% (v/v) goat serum for 30 min, cells wereincubated with primary following antibodies; anti-CD31 (Santa Cruz),anti-CD144 (ebioscience), anti-NRP-1 (Santa Cruz) and anti-a-SMA,(Chemicon) overnight at 4° C. Cells were washed with PBS, then incubatedwith secondary antibodies conjugated with Alexa-488 or Alexa-565(Molecular Probe) and visualized by confocal microscopy aftercounterstaining with 2 g/ml DAPI (Sigma-Aldrich). The confocal imageswere obtained with an Olympus FV1000 mpE confocal microscope using as anOlympus uplanSApo 60×W/1.2 NA/eus objective. All the images were takenas Z-stacks with individual 10μ thick sections at room temperature andimages were analyzed using FV10-ASW 3.0 Viewer.

Mice: All animal procedures were carried in accordance with theGuidelines for the Care and Use of Laboratory Animals and were approvedby the Institutional Animal Care and Use Committees (IACUCs) at IndianaUniversity School of Medicine (IACUC protocol #10850). Both male andfemale 6-12 week old NOD/SCID mice (T- and B-cell deficient, impairedcomplement) were used for all animal studies. NOD-SCID mice weremaintained under specific-pathogen-free conditions at the IndianaUniversity Laboratory Animal Resource Center (LARC). Previous work withthis animal model was used to determine the minimum number of animalsneeded to obtain statistically significant results (Yoder et al., 2007).Previous studies have shown that 8 out of 10 matrices (one animalreceived two matrices) implanted inosculate with the host vasculatureand that 8 matrices (4 animals) with functional vessels are needed foreach group for statistical significance (Yoder et al., 2007). Method ofrandomization was not used while allocating samples and animals to eachexperimental group. Also, investigator was not blinded to the groupallocation both during the experiment and when accessing the outcomes.

In vivo implantation/vessel formation assay (including functionalassay): Pig skin type I collagen was used to generate three-dimensional(3D) cellularized collagen matrices as previously described (Critser etal., 2010). Briefly, type 1 collagen gel mixture was prepared by mixingtogether ice-cold porcine skin collagen solution, 10% v/v human plateletlysate in 0.01N HCL, and neutralized with phosphate buffered saline and0.1 N NaOH to achieve neutral pH (7.4). Neutralized gel mixtures (˜1.5mg/mL) were kept on ice before induction of polymerization by warming at37° C., in 5% CO₂. KNA⁺ mesoderm cells or SSEA−KNA+ mesoderm cells orSSEA−KNA+-derived NRP-1⁺CD31⁺ ECFCs were added to the collagen mixtureto a final concentration of four million cells/ml collagen. The collagenmixture (250 μL) containing the cell suspension was added to 48-welltissue culture dishes and was allowed to polymerize to form gels byincubation in a CO₂ incubator at 37° C. for 30 minutes. The gels werethen overlaid with 500 μl of culture medium for 30 min at 37° C., in 5%CO₂. After 1 hour of ex vivo 3D culture, cellularized gels wereimplanted into the flanks (a bluntly dissected subcutaneous pouch ofanterior abdominal wall with close proximity of host vasculature) of 6-to 12-week-old NOD/SCID mice as previously described{Yoder, 2007 #71}.Surgical procedures to implant collagen gels were conducted underanesthesia and constant supply of oxygen. Incisions were sutured andmice monitored for recovery. Various days after implantation, gels wererecovered by excising engrafts in animals that had been humanelysacrificed per approved IACUC protocol. Confocal fluoresce imaging andimmunohistochemistry was performed as described previously using H&E andanti-human CD31 (and NRP-1) staining to examine the gels for humanendothelial-lined vessels perfused with mouse red blood cells. hCD31⁺blood vessels were imaged from each explant using a Leica DM 4000Bmicroscope (Leica Microsystems) with attached Spot-KE digital camera(Diagnostic Instruments). Functional vessels were counted only if theycontained at least 1 mouse erythrocyte. Olympus-FV-1000 MPE invertedconfocal/2P system us utilized to examine NRP-1⁺CD31⁺ vessels.

Gene and miRNA expression analysis: Reverse transcriptase (RT) reactionswere performed in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems).mRNA RT reactions were performed using Transcriptor Universal cDNAMaster (Roche). Specific miRNA primers were used for specific miRNA ofinterest for generating cDNA. RT reactions without templates or primerwere used as controls. Gene and miRNA expression levels were quantifiedusing the ABI 7300 RT-PCR System (Applied Biosytems). Quantitative PCRfor mRNA was performed using FastStart Universal SYBR green master (Rox)(Roche). Comparative real-time PCR with or without specific primers formiRNAs and mRNAs was performed in triplicates. mRNA reactions wereperformed at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 sand 60° C. for 1 min. miRNA reactions were performed by 40 cycles of 95°C. for 15 s and 60° C. for 1 min. Relative expression levels werecalculated using the comparative Ct method (Lee et al., 2013).

ECFC single cell proliferation assays: KNA+ or SSEA5−KNA+ mesodermcell-derived ECFCs were subjected to a single cell assay to evaluateclonogenic proliferative potential. Briefly, endothelial cells weretreated with trypLE Express (Invitrogen) to obtain a single cellsuspension. Cell counts and serial dilutions were performed to obtain aconcentration of 0.68 cells per well in individual wells of 96-wellculture plates. Wells were examined the day after plating to ensure thepresence of a single cell per well. Culture media was changed on days 4,8, and 12. On day 14 of culture, cells were stained with Sytox reagent(Invitrogen), and each well was examined to quantitate the number ofcells using a fluorescent microscope. Those wells containing two or morecells were identified as positive for proliferation under a fluorescentmicroscope at 10× magnification using a Zeiss Axiovert 25 CFL invertedmicroscope with a 10×CP-ACHROMAT/0.12 NA objective. Wells withendothelial cell counts of 2-50, 51-500, 501-2000 and ≥2001 were labeledas endothelial cell clusters (ECCs), low proliferative potential ECFCs(LPP) and high proliferative potential ECFCs (HPP) as previouslydescribed (Yoder et al., 2007; Ingram et al., 2005).

Western blot analysis: Cell lysates were prepared by resuspending cellsin lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1%Triton X-100, 2 mM EDTA, 1 mM Na₃VO₄, 1 μg/ml each of aprotinin andleupeptin) followed by incubation on ice for 20 min. Insolublecomponents were removed by centrifugation at 12,000×g for 15 min.Protein concentrations were determined with a protein assay kit(Bio-rad). Proteins were separated by electrophoresis on 4-20%Tris-glycine minigels and then transferred onto immobilon-FL PVDFmembrane (Millipore). Nonspecific binding was blocked with blockingbuffer for 1 hr at room temperature and incubated overnight at 4° C.with primary antibodies against phospho-PYK2 (1:1,000; Cell Signaling)and phospho-p130^(Cas) (1:1,000; Cell Signaling) in Odyssey blockingbuffer. Blots were washed with PBS containing 0.1% Tween20, followed byincubation for 1 hour at room temperature with anti-rabbit antibody(1:10,000; LI-COR). Immunoreactive bands were detected using the OdysseyInfrared Imager (LI-COR).

Fc-NRP-1 treatment assay: After 2 days (−D2) of culture of hiPSC clumpsin mTeSR1 media, cultures were directed toward the mesodermal lineagewith addition of activin A (10 ng/mL) in the presence of FGF-2, VEGF₁₆S,and BMP4 (10 ng/mL) for 24 hrs. The following day (D1), activin-Acontaining media was removed and replaced with 8 mL of Stemline IIcomplete media (Sigma) containing FGF-2 (Stemgent), VEGF₁₆₅ (R&D) andBMP4 (R&D) and 500 ng/mL of Fc-NRP-1 (R&D). Media was replaced with 8 mlof fresh Stemline II differentiation media containing FGF-2 (Stemgent),VEGF₁₆₅ (R&D) and BMP4 (R&D) and 500 ng/mL of Fc-NRP-1 (R&D) on day 3.On day 4 the cells were collected for sorting by flow cytometry forSSEA5⁻KNA+ mesoderm cells.

miRNA microarray and RNA sequence analysis: Total RNA was isolated fromthe samples using Trizol reagent (Invitrogen) and the RNA quality wasexamined as previously described (Ginsberg et al., 2012, Cell151:559-575). For miRNA microarray, miRNome miRNA PCR Array plates,miScript II Reverse Transcription reaction kits, and miScript SYBR GreenPCR Kits (all from Qiagen) were used to examine the expression profilesof the 1008 most abundantly expressed and best characterized miRNAsequences in the human miRNA genome (miRNome) as annotated in miRBaseRelease 16. For, RNA-seq analysis, RNA sequence library was generatedusing 1 μg of high quality total RNA and sequencing was performed usingIllumina HiSeq2000 sequencer as previously described (Ginsberg et al.,2012). The resulting sequence reads were mapped to the human genome(hg18) using TopHat with default parameters, and the RefSeq (June 2010)transcript levels (FPKMs) were quantified using CuffLinks.

miRNA mimic/inhibitor treatment assay: After 1 day (−D1) of culture ofsingle cell suspension of hiPSCs in mTeSR1 media, cultures were directedtoward the mesodermal lineage with addition of activin A (10 ng/mL) inthe presence of FGF-2, VEGF₁₆₅, and BMP4 (10 ng/mL) and 2.5 μg mimic orinhibitor/100,000 seeded cells/well of 6 well plate (GE Dharacon) for 24hrs. The following day (D1), activin-A and miRNA mimic or inhibitorcontaining media was removed and replaced with 2 mL of Stemline IIcomplete media (Sigma) containing FGF-2 (Stemgent), VEGF₁₆₅ (R&D) andBMP4 (R&D). Media was replaced with 2 ml of fresh Stemline IIdifferentiation media containing FGF-2 (Stemgent), VEGF₁₆₅ (R&D) andBMP4 (R&D) and 2.5 μg mimic or inhibitor on day 2. Media was replacedwith 2 ml of fresh Stemline II differentiation media containing FGF-2(Stemgent), VEGF₁₆₅ (R&D) and BMP4 (R&D) on day 3. On day 4 the cellswere collected for sorting by flow cytometry for SSEA5−KNA+ mesodermcells.

Statistical analysis: All experiments were performed ≥3 times intriplicate and data are represented as mean value ±SD for statisticalcomparison. A power of analysis with a 95% confidence interval was usedto calculate sample size required to obtain statistically significantresults. The sampling number we used gave a normal distribution.Significance of differences was assessed by a two tailed student'st-test or one way ANOVA-Tukey post-hoc test Multiple Comparison Test.

Example 2

Referring now to FIGS. 1A-1G, NCAM and APLNR co-expressing cells withinday 4 (D4) KDR⁺ mesoderm cells (KNA⁺ mesoderm) gave rise to NRP-1⁺CD31⁺endothelial cells with ECFC competence.

Human PSCs cultured in mTeSR1 were induced to differentiate intomesoderm cells under 2D, serum and feeder-free conditions. PSCs werecultured in Stemline-II medium with FGF-2 (10 ng/mL), BMP4 (20 ng/mL),VEGF165 (10 ng/mL) and Activin-A (10 ng/mL) for 24 hours (i.e., fromDO-D1) (FIG. 1A). On D1, the cell culture medium was replaced withStemline-II medium with FGF-2 (10 ng/mL), BMP4 (20 ng/mL), VEGF₁₆₅ (10ng/mL). This replacement medium was used to culture the cells undergoingmesodermal induction for 3 days (i.e., from D1-D4).

On Day 4, the cells induced to differentiate into mesoderm cells weresorted (FIG. 1B). KDR⁺ cells were gated for NCAM and APLNR expression.KDR⁺NCAM⁺APLNR⁺ (K⁺N⁺A⁺, also referred to herein as KNA⁺) andKDR⁺NCAM⁺APLNR⁻ (K⁺N⁺A⁻) KDR⁺NCAM⁻APLNR⁻ (K⁺N⁻A⁻) cells were sorted forfurther differentiation and examination for the emergence of NRP-1⁺CD31⁺ECFC-like cells (FIG. 1C). Sorted K⁺N⁺A⁺, K⁺N⁺A⁻ and K⁺N⁻A⁻ mesodermsub-sets that were further differentiated into ECFC lineage for another9 days (3 plus 9, total of 12 days) to examine for the emergence ofNRP-1⁺CD31⁺ cells at various days of differentiation (FIG. 1D). At day12, the K⁺N⁺A⁺ mesoderm fraction gave rise to NRP1⁺CD31⁺ cells thatformed a homogenous cobblestone endothelial monolayer, displayed uniformco-expression for CD31 and CD144 endothelial markers and completelylacked α-SMA expression (top panels of FIG. 1D), suggesting a stableECFC-like phenotype. However, cells isolated from the other two subsets(i.e., K⁺N⁺A⁻ (center panels of FIG. 1D) and K⁺N⁻A⁻ (lower panels ofFIG. 1D)) lacked adequate NRP-1 expression, formed heterogeneous cellmonolayers, displayed expression for CD144 but lacked uniformco-expression for CD31 and CD144 endothelial markers and exhibitedexpression for the non-endothelial marker α-SMA, suggesting a completelack of a stable ECFC phenotype. The K⁺N⁺A⁺ mesoderm-derived NRP-1⁺CD31⁺cells exhibited high clonal proliferative potential with a hierarchy ofcolonies ranging from clusters of 2-50 cells up to colonies of >2001similar to that of hiPSC-ECFC-like cells (FIG. 1E). However, cellsisolated from the other two subsets (i.e., K⁺N⁺A⁻ and K⁺N⁻A⁻) failed toexhibit high clonal proliferative potential. K⁺N⁺A⁺ mesoderm-derivedNRP-1⁺CD31⁺ cells produced robust in vivo human blood vessels filledwith host murine red blood cells (FIG. 1F, top right panel; arrows pointto blood vessels) similar to those produced by hiPSC-ECFC-like cells(FIG. 1F, top left panel). However, cells isolated from the other twosubsets (i.e., K⁺N⁺A⁻ and K⁺N⁻A⁻) failed to produce robust in vivo humanblood vessels filled with host murine red blood cells (FIG. 1F, bottomleft and right panels, respectfully; arrows point to hCD31⁺ functionalblood vessels. Similarly, functional hCD31⁺ blood vessels were generatedby K⁺N⁺A⁺ mesoderm-derived NRP-1⁺CD31⁺ cells hiPSC-ECFC-like cells, butnot by K⁺N⁺A⁻ or K⁺N⁻A⁻ mesoderm cells (FIG. 1G).

Referring now to FIGS. 2A-2C, human iPS cells after 3-5 days ofdifferentiation using the above culture protocol generate cellsexpressing mesoderm markers and lacking typical endothelial surfaceexpression. APLNR⁺ cells were found only in the KDR⁺NCAM⁺ mesodermsub-set and were absent in KDR⁻NCAM⁻ sub-set (FIG. 2A) and KDR⁺ cellswere found at highest levels at day 3 and 4 and decreased over time(FIG. 2B). Typical endothelial marker (CD31, NRP-1 and CD144) expressionwas not found in any day 4 differentiated cells (FIG. 2C).

Referring now to FIGS. 3A-3B, direct in vivo differentiation of day 4KNA⁺ mesoderm cells that were isolated without selecting against SSEA5+cells formed robust human blood vessels, but also producedendoderm-derived cell-like derivatives.

KDR⁺ cells were gated for NCAM and APLNR expression. APLNR⁺ andAPLNR-mesoderm cells were sorted for further direct in vivo implantationand examination (FIG. 3A). Sorted APLNR⁻ mesoderm cells producedteratomas after 2 months of in vivo implantation (FIG. 3B, left panel).The APLNR⁺ mesoderm sub-set (FIG. 3B, right panel) formed robust in vivohuman blood vessels filled with host murine red blood cells (blue openarrows) with accompanying endoderm-derived cell like derivatives (pinkclosed arrows).

Referring now to FIGS. 4A-4D, direct in vivo differentiation of D4 SSEA5depleted KNA⁺ mesoderm cells (i.e., SSEA5⁻KDR⁺NCAM⁺APLNR⁺, also referredto as SSEA5⁻KNA⁺) formed robust human blood vessels without giving riseto teratoma or endoderm-derived cell-like derivatives.

Day 4 differentiated hiPSCs were first gated for SSEA5 and KDRexpression (FIG. 4A, left). The SSEA5⁻KDR⁺ cells were gated for NCAM andAPLNR expression (FIG. 4A, right). SSEA5⁻KDR⁺NCAM⁺APLNR⁺ (SSEA5⁻KNA⁺)and SSEA5⁻KDR⁺NCAM⁺APLNR⁻ (SSEA5⁻KNA⁻) cells were sorted for furtheranalysis. SSEA5⁻KNA⁺ cells formed robust functional in vivo vessels(FIG. 4B, blue arrows, left panel), SSEA5⁻KNA⁻ cells failed to formrobust in vivo vessels (FIG. 4B, white arrows, right panel). Similarly,functional hCD31⁺ blood vessels were generated by SSEA5⁻KNA⁺ cells, butnot by SSEA5⁻KNA⁻ cells (FIG. 4C). When implanted in vivo, SSEA5⁻KNA⁺cells formed NRP-1⁺CD31⁺ ECFC vessels as early as 8 days afterimplantation (FIG. 4D).

Referring now to FIGS. 5A-5C, day 4 SSEA5 depleted KNA⁺ mesoderm cellsdisplayed transcripts typically enriched in lateralplate/extra-embryonic mesoderm cells, and exhibited enhanced formationof NRP-1⁺CD31⁺ cells with ECFC competence upon in vitro ECFCdifferentiation.

Gene expression analysis revealed that SSEA5⁻KNA⁺ cells over-expressedlateral plate-extra-embryonic mesoderm markers, relative to hiPSCs, andlacked expression of axial, paraxial and intermediate mesoderm markers(FIG. 5A). Sorted SSEA5⁻KNA⁺ cells were further differentiated into theECFC-like lineage for another 8 days (4 plus 8, total of 12 days). Atday 12, SSEA5⁻KNA⁺ cells produced ≥3 fold more NRP-1⁺CD31⁺ cellscompared to NRP-1⁺CD31⁺ cells produced from hiPSCs induced todifferentiate into an ECFC-like lineage (i.e., 12 days differentiationwithout isolating a SSEA5⁻KNA⁺ mesoderm sub-set at day 4 ofdifferentiation) (FIG. 5B; left panel, bar graph). NRP1⁺CD31⁺ cellsderived from the SSEA5⁻KNA⁺ mesoderm cells formed a homogenouscobblestone endothelial monolayer (FIG. 5B, left middle panel),displayed uniform co-expression for CD31 and CD144 endothelial markers(FIG. 5B, right panel) and completely lacked α-SMA expression (FIG. 5B,right middle panel), suggesting that the SSEA5⁻KNA⁺ mesoderm cells wereable to differentiate into a stable ECFC-like phenotype. SSEA5⁻KNA⁺mesoderm-derived NRP-1⁺CD31⁺ cells exhibited high clonal proliferativepotential with a hierarchy of colonies ranging from clusters of 2-50cells up to colonies of >2001 similar to that of hiPSC-ECFCs (FIG. 5C).However, cells isolated from SSEA5⁻KNA⁻ mesoderm sub-set failed toexhibit high clonal proliferative potential (FIG. 5C).

Referring now to FIGS. 6A-6G, the inventors found that Fc-NRP-1 mediatesVEGF-KDR signaling through p130^(Cas)/Pyk2 activation and enhancesformation of SSEA5⁻KNA⁺ mesoderm cells from hiPSCs.

NRP-1, KDR, Fc-NRP-1, NRP-1-B and VEGF₁₆₅ functions in endothelial cellsare shown in FIG. 6A. Briefly, NRP-1 functions as a VEGF₁₆₅ co-receptorand brings VEGF₁₆₅ to its receptor (KDR). Fc-NRP-1 acts as surrogate formembrane NRP-1 and binds and brings VEGF₁₆₅ to KDR. In contrast, NRP-1-Bblocking antibody selectively binds to the VEGF₁₆₅ binding site ofNRP-1, thereby specifically blocking binding of VEGF₁₆₅ to NRP-1.

Examination of KDR and p130^(Cas)/Pyk2 phosphorylation was carried outby Western blotting (FIGS. 6B and 6C). KDR phosphorylation was observedin VEGF stimulated groups and Fc-NRP-1 dimer treatment increasedphosphorylation of KDR compared to control treated cells. However,decreased phosphorylation was observed in NRP-1-B treated cells.Similarly, increased p130^(Cas)/Pyk2 phosphorylation was observed inFc-NRP-1 dimer treated cells compared to NRP-1-B treated cells.

Gene expression of VEGF-A isoforms in hiPSCs and SSEA5⁻KNA⁺ mesodermcells was investigated. VEGF-A isoforms were not up-regulated inSSEA5⁻KNA⁺ mesoderm cells compared to hiPSCs (FIGS. 6D and 6E).

A mesoderm lineage differentiation protocol that involves culturing thecells undergoing mesoderm induction in the presence of Fc-NRP-1 dimerand growth factors is shown in FIG. 6F. Dimeric Fc-NRP-1 treatment todifferentiating hiPSCs caused more than a 2-fold increase in theproduction of SSEA5⁻KNA⁺ mesoderm cells compared to Fc-NRP-1-untreatedcells (FIG. 6G).

Referring now to FIGS. 7A-7E, specific miRNA mimics miRNA inhibitors andcombinations thereof enhance SSEA5⁻KNA⁺ mesoderm formation from hiPSCs.

In order to identify miRNAs and their putative transcription factortargets relevant to SSEA5⁻KNA⁺ mesoderm formation, we performed miRNAmicro-array and RNA-seq analysis of Day 0 undifferentiated hiPSCs andDay 4 SSEA5⁻KNA⁺ mesoderm cells (FIG. 7A). Our analysis of expression ofmiRNA and mRNA transcripts revealed 12 miRs (miR-221-3p, miR-1271-5p,miR-559, miR-543, miR-361-3p, miR-30d-5p, miR-124-3p, miR-185-5p,miR-330-5p, miR-145-5p, miR-214-3p and miR-497-5p) with published andvalidated targets that potentially regulate transcription factorsrelevant to SSEA5⁻KNA⁺ mesoderm formation from hiPSCs. Among these 12candidate miRNAs, we found 8 miRNAs (miR-221-3p, miR-1271-5p, miR-559,miR-543, miR-361-3p, miR-30d-5p, miR-124-3p and miR-185-5p) wereexpressed at lower levels in Day 4 SSEA5⁻KNA⁺ mesoderm cells compared toDay 0 undifferentiated hiPSCs, and four miRNAs (miR-330-5p, miR-145-5p,miR-214-3p and miR-497-5p) were expressed at higher levels in Day 4SSEA5⁻KNA⁺ mesoderm cells compared to Day 0 undifferentiated hiPSCs.

To determine the effect of mimicking and/or inhibiting the identifiedmiRNAs on mesoderm lineage differentiation, we developed a protocol forinducing mesoderm generation from hiPSCs in the presence of specificmiRNA mimics, miRNA inhibitors, both mimics and inhibitors (3m3i), ormimic/inhibitor controls (FIG. 7B). Addition of specific mimics for 3miRNAs (miR-330-5p, miR-145-5p and miR-214-3p), specific inhibitors for3 miRNAs (miR-221-3p, miR-1271-5p, miR-559) or 3 mimics and 3 inhibitorscombined increased the frequency of SSEA5⁻KNA⁺ mesoderm cells comparedto the control (FIG. 7C). Addition of these specific mimics, inhibitors,or 3m3i decreased the expression of most of the miRNAs that wereidentified (in our miRNA array analysis) to be expressed at lower levelsin Day 4 SSEA5⁻KNA⁺ mesoderm cells (FIG. 7D). Addition of these specificmimics, inhibitors or 3m3i increased the expression of some of themiRNAs that were identified (in our miRNA array analysis) to beexpressed at higher levels in Day 4 SSEA5⁻KNA⁺ mesoderm cells (FIG. 7E).

Referring now to FIGS. 8A-8D, miR-214-3p targets CLDN6 indifferentiating hiPSC and enhances formation of SSEA5⁻KNA⁺ mesodermcells.

Expression of miR-214 and its putative target CLDN6 was analyzed inhiPSCs and SSEA5⁻KNA⁺ mesoderm cells. We found that miR-214 and CLDN6exhibit an inverse expression correlation in hiPSCs and SSEA5⁻KNA⁺mesoderm cells, respectively (FIG. 8A). miR-214 is highly expressed inSSEA5⁻KNA⁺ cells compared to hiPSCs (FIG. 8A, left bar) and CLDN6 isexpressed at lower levels in SSEA5⁻KNA⁺ cells compared to hiPSCs (FIG.8A, right bar). Over-expression of miR-214 in differentiating hiPSCscaused more than a 2-fold increase in the production of SSEA5⁻KNA⁺mesoderm cells compared to a GFP reporter control vector (FIG. 8B). Aluciferase reporter assay confirmed that miR-214 directly regulatesexpression of wild type (WT) CLND6 (FIG. 8C). Differential geneexpression analysis between hiPSC and SSEA5⁻KNA⁺ mesoderm cells revealedthat CLND6 is one of the most down-regulated genes among the 12validated miR-214 targets that decreased in the SSEA5⁻KNA⁺ mesoderm(FIG. 8D). For example, CLDN6 is only expressed at only 4% in theSSEA5⁻KNA⁺ mesoderm cells compared to the level measured in the hiPSCs.

Although the disclosure has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the purpose and scope ofthe disclosure as outlined in the claims appended hereto. Any examplesprovided herein are included solely for the purpose of illustrating thedisclosure and are not intended to limit the disclosure in any way. Anydrawings provided herein are solely for the purpose of illustratingvarious aspects of the disclosure and are not intended to be drawn toscale or to limit the disclosure in any way. The disclosures of allprior art recited herein are incorporated herein by reference in theirentirety.

1-14. (canceled)
 15. The method of claim 22, wherein the endothelialinducing step is carried out in the absence of one or more of:co-culture cells, embryoid body formation and exogenous TGF-βinhibition.
 16. The method of claim 22, wherein the isolated ECFC-likecells have a capacity to form blood vessels when implanted into a mammalin the absence of co-implanted cells.
 17. An isolated population ofhuman KDR⁺NCAM⁺APLNR⁺ mesoderm cells, wherein the isolatedKDR⁺NCAM⁺APLNR⁺ mesoderm cells have a capacity to form blood vesselswhen implanted into a mammal and wherein the isolated KDR⁺NCAM⁺APLNR⁺mesoderm cells were derived in vitro from human pluripotent stem cellsin a method carried out in the absence of co-culture cells.
 18. Theisolated population of claim 17, wherein the KDR⁺NCAM⁺APLNR⁺ mesodermcells are SSEA5⁻.
 19. The isolated population of claim 17, wherein theKDR⁺NCAM⁺APLNR⁺ mesoderm cells exhibit increased expression of one ormore lateral plate-extra-embryonic mesoderm markers relative to PSCs,wherein the lateral plate-extra-embryonic mesoderm markers are selectedfrom BMP4, WNT5A, NKX2-5 and HAND1.
 20. The isolated population of claim19, wherein the KDR⁺NCAM⁺APLNR⁺ mesoderm cells lack increased expressionof one or more axial mesoderm markers, paraxial mesoderm markers and/orintermediate mesoderm markers, relative to PSCs, wherein the axialmesoderm markers are selected from CHIRD and SHH, the paraxial mesodermmarkers are selected from PAX1, MEOX1, and TCF15, and the intermediatemesoderm markers are selected from GOSR1, PAX2 and PAX8.
 21. An isolatedpopulation of human KDR⁺NCAM⁺APLNR⁺ mesoderm cells obtained according toa method comprising: (a) providing pluripotent stem cells (PSCs): (b)inducing the pluripotent stem cells to undergo mesodermaldifferentiation, wherein the mesodermal induction is carried out in theabsence of co-culture cells and further comprises: i) culturing thepluripotent stem cells for 24 hours in a mesoderm differentiation mediumcomprising Activin A, bone morphogenetic protein-4 (BMP-4), vascularendothelial growth factor (VEGF) and fibroblast growth factor (FGF-2);ii) replacing the medium of step i) with a mesoderm differentiationmedium comprising BMP-4, VEGF and FGF-2 every 24-48 hours thereafter for72 hours; and (c) isolating from the cells induced to undergodifferentiation to mesoderm cells, wherein the isolation of mesodermcells comprises: iii) sorting the mesoderm cells to select forKDR+NCAM+APLNR+ cells.
 22. An isolated population of human NRP-1+CD31+endothelial colony forming cell-like cells (ECFC-like cells) obtainedaccording to a method comprising generating an isolated population ofhuman KDR+NCAM+APLNR+ mesoderm cells, wherein the generation of humanKDR+NCAM+APLNR+ mesoderm cells population comprises: (a) providingpluripotent stem cells (PSCs); (b) inducing the pluripotent stem cellsto undergo mesodermal differentiation, wherein the mesodermal inductioncomprises: i) culturing the pluripotent stem cells for 24 hours in amesoderm differentiation medium comprising Activin A, BMP-4, VEGF andFGF-2; ii) replacing the medium of step i) with a mesodermdifferentiation medium comprising BMP-4, VEGF and FGF-2 every 24-48hours thereafter for 72 hours; and (c) isolating from the cells inducedto undergo differentiation the mesoderm cells, wherein the isolation ofmesoderm cells comprises: iii) sorting the mesoderm cells to select forKDR+NCAM+APLNR+ cells; wherein the method further comprising inducingthe said population of mesoderm cells to undergo endothelialdifferentiation, wherein the induction of endothelial differentiationcomprises: (d) culturing the isolated mesoderm cells in an endothelialdifferentiation medium comprising BMP-4, VEGF and FGF-2 for 6-8 days;and (e) isolating from the cells induced to undergo endothelialdifferentiation endothelial colony forming cell-like (ECFC-like) cells,wherein the ECFC-like cells are CD31+ NRP-1+ and exhibit a cobblestonemorphology.
 23. A pharmaceutical composition comprising theKDR⁺NCAM⁺APLNR⁺ mesoderm cells of claim
 17. 24. A pharmaceuticalcomposition comprising the endothelial colony forming cell-like cells(ECFC-like cells) of claim
 22. 25. A method of examining a test agentfor its ability to modify cellular activity, the method comprising:exposing at least one of the cells of the population of cells of claim17 to a test agent and; observing the effect of the test agent on one ormore of cell growth and cell viability.
 26. A method for transplantationin a subject in need thereof, the method comprising: providing to thesubject the isolated population of cells of claim
 17. 27. A method oftreating a subject in need of epithelial repair, the method comprising:providing to the subject a therapeutically effective amount of apopulation of cells according to claim 17.